I did a cell bio lab recently where we counted fixed cells using a hemocytometer. It was straightforward, but I got some wacky results. We counted cells for a control, dilute, and concentrated sample. All of these came from the same initial fixed stock. We also did a differential count for dividing cells using the same samples

My weird results showed up between my control and my concentrated samples. Since my cells were fixed, I expected to have the same ratios between all samples for the dividing cells.

To prepare the concentrated solution, we centrifuged cells, and then we were supposed to pour off part of the supernatant. However, my TA poured it all off. She then added Tris to it to make it the correct volume.

My question is this:What is tris, and what does it do. Would it have any effect on the cell population? Does it lyse cells? Could this have caused weird results with dividing cells?

Tris is just a buffer http://en.wikipedia.org/wiki/Tris it does not lyse cells. It's basically an environment the cells are happy in because it has the right pH (your TA probably used Trist pH=7.4).

The anomaly you witnessed was probably due to the fact that the hemocytometer is not as precise when the sample is really concentrated, and the thing has an incredibly high error rate anyway (which is why biologists don't really use the thing anymore, there are special devices that can count cells much more accurately).

"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

I use hemocytometer every week! :P Though I wish we bough an automated cell counter, but hey, as long as humans can do it (seemingly) for free, why spend money on things?!

Anyway, yeah the damn thing has a high error rate on both low and high concentrations (and pretty high error rate even in the between), because almost always there's some element like pipetting error, cell sedimentation, counting error, bad moon phase or all those combined that affect the outcome.

That being said, it does give a good rough estimation of cell numbers, which is often well enough. Also, if you do it a lot, you standardize your techniques, and you have the patience to count enough of cells, you can get quite accurate numbers as well. Just don't go and compare your results with your lab tech: you'll notice that she'll probably get a completely different number, and since she's done cell counting since you were born, she's probably right and you notice your counting sucks!

you don't do it for free, a researcher's time is valuable. that's why devices and automation in research exist, so whenever possible you can spend time doing creative experiments, not boring stuff that a monkey could do

"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

MrMistery wrote:you don't do it for free, a researcher's time is valuable. that's why devices and automation in research exist, so whenever possible you can spend time doing creative experiments, not boring stuff that a monkey could do

Well, that's the ideal situation, but you can bet it doesn't always go like that :)

In my earlier post I put the word "seemingly" in quotation marks to indicate that too often the people who decide on finances are happy as long as the figures look good: no big expendures, no big investments = good, even though this means that the people doing research spend their time on doing monkey tasks. Simply as long as the research papers keep coming they are quite happy, despite the fact that there could be more papers if we had, say, an automated cell counter. There is no price tag for me counting cells, in academia, so to the big bosses it seems as if there were no expenses, because me counting the cells does not show in any of their figures. Hence, haemocytometre = free (GOOD), automated cell counter = expensive (BAD).

Of course it all depends on how rich your given faculty/departemt/research group is, but in your average lab it goes pretty much like this: they have X money for the salaries and Z money for reagents and equipment. The X is just enough to keep the people there, and Z usually goes for stuff that are essential for research, like key reagents and maintenance of existing equipment. Thus they quite rarely have money for bigger investments, and thus you don't always get your cell counter. So, you'll just be the happy monkey you are ;)

Now, if I was doing this stuff in a private corporation, it might be different: they have long since figured out that time is money, and thus they can calculate these figures way better - I'd probably get my cell counter in no time. Then again, private corporations can also go to bankruptcy if they don't get these figures right, but universities can often keep going even if their people spent half their time on stuff a monkey could do!